Fibrr-shaping peptides capable of interacting with self-assembling peptides

ABSTRACT

The present invention relates to a fibre-shaping peptides that are capable of interacting with self-assembling peptides to form protein structures. The present invention also relates to methods of forming protein structures using the fibre-shaping peptides of the present invention.

The present invention relates to fibre-shaping peptides that are capableof interacting with self-assembling peptides to form protein structures.The present invention also relates to methods of forming proteinstructures using the fibre-shaping peptides of the present invention.

Biological assemblies provide inspiration for the development of newmaterials for a variety of applications (Holmes, Trends Biotechnol., 20,16-21, 2002 and Yeates et al., Curr. Opin. Struct. Biol., 12, 464-470,2002). The ability to realise this potential, however, is hampered bydifficulties in producing and engineering natural biomaterials, and indesigning them de novo. Recently, the inventors described aself-assembling system comprising two short, synthetic polypeptides(dubbed self-assembling peptides (also referred to as straights)herein), which combine to form extended fibres (see International PatentApplication WO 01/21646 and Pandya et al., Biochemistry, 39, 8728-8734,2000). The fibres described in WO 01/21646 are about 50 nm in diameter,and extend straight and without branching for tens to hundreds ofmicrons. It is desirable to influence and to control fibre morphology.

Previously, the inventors applied the concept of sticky-end directedmolecular assembly, which is well documented for the assembly of DNA, topeptides. This led to a self-assembling peptide fibre (SAF) system(Pandya et al., Biochemistry, 39, 8728-8734, 2000). The system comprisestwo short peptides (SAF-p1 and SAF-p2) of de novo design. The SAF-p1 andSAF-p2 sequences were based on accepted design principles for parallel,hetero-dimeric coiled coils, namely leucine zippers (Harbury et al.,Science, 262, 1401-1407, 1993; O'Shea et al., Curr. Biol., 3, 658-667,1993; Woolfson et al., Prot. Sci., 4, 1596-1607, 1995; Ciani et al., J.Biol. Chem., 277. 10150-10155, 2002). However, the SAF peptides wereeach designed with two distinct regions or subunits: A and B in SAF-p1and C and D in SAF-p2, respectively; where A complements D and Bcomplements C. Thus, as depicted in FIG. 1 a, co-assembly of the twopeptides should lead to sticky-ended hetero-dimers, which should furtherassemble into fibres (see FIG. 1 b). These design features wereconfirmed experimentally using a combination of spectroscopy, X-rayfibre diffraction and microscopy; although, interestingly, the fibreswere thicker than originally anticipated. For instance, transmissionelectron microscopy (TEM) revealed that, when mixed in water and allowedto mature for short periods, the SAF peptides produced linear structures40-50 nm in diameter that extended for many microns, FIG. 2 a. Exceptfor some rare examples where the fibres were bent, there was no evidencefor non-linear or branched structures. More recently, the inventorsredesigned SAF-p2 to make SAF-p2a, which combines with SAF-p1 moreefficiently and produces fibres with significantly improved stabilityand internal order compared with the original design; otherwise, theappearance of the matured fibres was not altered.

U.S. Pat. No. 5,229,490 and International Patent Application No. WO92/18528 are directed to branched peptides; however, the branchedpeptides do not interact with self-assembling peptides for form proteinstructures. The branched polypeptides are used to display antigens.

It would be of considerable interest to alter fibre size and morphologythrough rational design: for instance, such that specific fibres couldbe tailored and/or fibre assembly could be made to respond to patternedsurfaces, or to cultures of growing cells. One possibility is tointroduce special peptides (herein referred to as fibre-shapingpeptides) that complement and assemble with the straight SAF buildingblocks (self-assembling peptides), but introduce discontinuities intothe regularly repeating linear structure.

The present invention provides a fibre-shaping peptide comprising a huband a plurality of peptide monomer units each being attached at one endthereof to the hub, wherein the free ends of at least 2 peptide monomerunits are N-termini or C-termini, and each of the at least 2 peptidemonomer units is capable of interacting with a subunit of aself-assembling peptide to form an overlapping staggered structure.

The fibre-shaping peptides of the present invention allow morphologicalchanges to be made to protein fibres comprising self-assemblingpeptides. In particular, the fibre-shaping peptides allow one toincorporate branches, splits, kinks and bends in the protein fibres. Bybeing able to incorporate such morphological changes in the proteinfibres it is possible to generate a variety of protein structures, suchas assemblies in general, including matrices, filters, networks, grids,scaffolds, etc.

As indicated above, the fibre-shaping peptide of the present inventioncomprises a hub to which peptide monomer units are attached. The peptidemonomer units are attached covalently to the hub. The hub can be anymolecule which has at least 2 derivatisable sites (so that it ispossible to attach at least 2 peptide monomer units) and which does notprevent at least 2 peptide monomer units interacting with a sub-unit ofa self-assembling peptide to form an overlapping staggered structure. Itis further preferred that the hub has more than 2 derivatisable sitesenabling the attachment of more than 2 peptide monomer units and/or theattachment of one or more active molecules. It is particularly preferredthat the hub has 3 or 4 derivatisable groups.

In a preferred embodiment the fibre-shaping peptide comprises one ormore active molecules attached to the hub. The active molecule can beany molecule that has a desired function provided it does not prevent atleast 2 peptide monomer units interacting with a sub-unit of aself-assembling peptide to form an overlapping staggered structure.Suitable active molecules include an antibody molecule (i.e. amonoclonal antibody or functional part thereof, including Fab, Fv,F(ab′)₂ fragments and single chain Fv fragments), a receptor, a ligand,an enzyme, an antigen, a label, a metal ion or a nucleic acid molecule.In a particularly preferred embodiment the active molecule is biotin.The biotin molecule can be used to bind streptavidin, which may be freeor attached to a desirable molecule, such as a label or other activemolecules. The active molecule may be used to bind a desired substancefrom a solution. For example, an antibody molecule may be used to bindthe corresponding antigen. Alternatively, a receptor can be used to bindthe corresponding ligand. When the active molecule is a nucleic acid, itcan be used to bind transcription factors or even complementary nucleicacids. It is particularly preferred that the active molecule is an RGDbased peptide. The RGD peptide can be used to isolate cells from asolution.

The hub is preferably one or more amino acids, more preferably 1 to 6amino acids and most preferably 1 amino acid. In a preferred embodiment,the hub is lysine. When the hub is lysine, it is possible to attach 2peptide monomer units via their C-terminus ends to the amino groups. Afurther peptide monomer unit or an active molecule can be attached tothe carboxylic acid group.

In a further preferred embodiment the hub is glutamic acid. When the hubis glutamic acid, it is possible to attach 2 peptide monomer units viatheir N-terminus ends to the carboxylic acid groups. A further peptidemonomer unit or an active molecule can be attached to the amino group.

Preferably the peptide monomer units and the functional molecules arelinked to the hub via flexible linkers. The flexible linker may be anysuitable linker. Preferably the flexible linker is composed of aminoacids such as glycine, serine, alanine and O-alanine. It is particularlypreferred that the flexible linker is a poly-β-alanine peptidecomprising between 2 and 10 residues, more preferably about 3 to 5residues. The flexible linker assists in allowing the peptide monomerunits to easily interact with the self-assembling peptides and allowsany functional molecules to exert their function.

The term “a peptide monomer unit” as used herein refers to a peptidethat can interact with a sub-unit of a self-assembling peptide. In otherwords the peptide monomer unit is complementary to a sub-unit of aself-assembling peptide. The peptide monomer units of the fibre-shapingpeptide interact with self-assembling peptides to form overlappingstaggered structures which then self-assemble into a protein structureas described in WO 01/21646, and as shown in FIG. 1. The sub-unit of theself-assembling peptide is a region that specifically interacts with thepeptide monomer unit. Generally, the sub-unit is at one end of theself-assembling peptides so that on interaction an overlapping staggeredstructure is formed. Preferably the peptide monomer units of thefibre-shaping peptide and the self-assembling peptides comprise a heptadand/or a hendecad repeat motif. It is also preferred that at least oneof the peptide monomer units comprises an amino acid residue which iscomplementary to a residue in a sub-unit of the self-assembling peptideto encourage the fibre-shaping peptide and the self-assembling peptideto form a staggered parallel heterodimer. The amino acid residue in thepeptide monomer unit may be any residue which can pair with acomplementary amino acid in the sub-unit of the self-assembling peptide.Preferably the complementary amino acids are pairs of asparagines,arginines or lysines. It is also preferred that the complementary aminoacids are at interfacial sites on the peptides. Preferably thecomplementary amino acids are in the “a” position within the heptad orhendecad repeat motif on the peptide monomer unit and in the sub-unit ofthe self-assembling peptide.

The fibre-shaping peptide of the present invention may comprise morethan 2 peptide monomer units. It is preferred that the fibre-shapingpeptide comprises 2 to 10 peptide monomer units, more preferably 2 to 5peptide monomer units, most preferably 2 peptide monomer units. Asindicated above, the number of peptide monomer units in thefibre-shaping peptide will depend on the number of derivatisable groupson the hub.

As indicated above, at least 2 of the peptide monomer units must haveeither free N-terminal ends or free C-terminal ends. By ensuring thatthe fibre-shaping peptide comprises 2 peptide monomer units having thesame free ends (i.e. both C-terminal or both N-terminal ends), 2self-assembling peptides are forced to converge leading to adiscontinuity in the protein structure formed by the self-assemblingpeptides (see FIGS. 1 c and d).

The term “self-asssembling peptide” as used herein refers to a peptidethat can interact with other self-assembling peptides to form asubstantially linear structure, preferably a straight protein fibre. Theself-assembling peptides preferably associate in a parallel andcontiguous manner. Suitable self-assembling peptides are described in WO01/21646. Preferably the self-assembling peptide comprises a heptad orhendecad repeat motif, wherein a pair of complementary amino acidsresidues on different self-assembling peptides encourage theself-assembling peptides to form a staggered parallel heterodimercoiled-coil. The complementary amino acid residues may be any residueswhich can form a pair. Preferably the complementary amino acids arepairs of asparagines, arginines or lysines. It is also preferred thatthe complementary amino acids are at interfacial sites on the peptides.Preferably the complementary amino acids are in the “a” position withinthe heptad or hendecad repeat motif in the self-assembling peptide.

It is particularly preferred that the self-assembling peptide has thesequence NH₃-KIAALKQKIASLKQEIDALEYENDALEQ-COOH (SAF-p1) or the sequenceNH₃-KIRRLKQKNARLKQEIAALEYEIAALEQ-COOH (SAF-p2a).

The present invention also provides a self-assembling peptide having thesequence NH₃-KIRRLKQKNARLKQEIAALEYEIAALEQ-COOH (SAF-p2a).

The standard single letter amino acid terminology is used in thesequences given in the present application.

The term “overhanging staggered structure” refers to a structure inwhich 2 peptides assemble to form a heterodimer having overhanging endsthat are not interacting within the heterodimer.

The peptides of the present invention, including the fibre-shapingpeptides and the self-assembling peptides are preferably between 15 and100 amino acids in length, more preferably between 20 and 50 amino acidsin length, most preferably about 30 amino acids in length. The peptidesmay comprise naturally occurring amino acids, synthetic amino acids andnaturally occurring amino acids that have been modified.

The term “fibre” as used herein refers to a protein structure assembledfrom overlapping staggered structures interacting through theoverhanging ends. A number of fibres may interact laterally therebyforming thicker fibres. It is particularly preferred that the termrefers to a hetero-dimeric coiled coil structure.

The term “amino acid” as used herein refers to naturally occurring aminoacids, synthetic amino acids and naturally occurring amino acids thathave been modified.

In a preferred embodiment of the present invention the fibre-shapingpeptide of the present invention has the formula:(NH₃-g(abcdefg)_(q)abcde-(X)_(m))_(n)—Y—((X)_(m)-Z)_(p)  (1)or(Z-(X)_(m))_(p)—Y—((X)_(m)-g(abcdefg)_(q)abcdef-COOH)_(n)  (II)wherein abcdefg is a heptad repeat motif;X is a flexible linker;Y is a hub;Z is a functional molecule;_(q) is 1 to 15;_(m) is 0 or 3;_(n) is 2 to 10; and_(p) is 0 to 4.

In a particularly preferred embodiment of the present invention thefibre-shaping peptide of the present invention has the formula:(NH₃-g(abcdefg)_(q)abcde-(X)_(m))_(n)—Y—((X)_(m)-Z)_(p)  (I)or(Z-(X)_(m))_(p)—Y—((X)_(m)-g(abcdefg)_(q)abcdef-COOH)_(n)  (II)wherein abcdefg is a heptad repeat motif;X is a flexible linker;Y is a hub;Z is a functional molecule;_(q) is 1 to 15;_(m) is 0 or 1;_(n) is 2 to 10; and_(p) is 0 to 4.

Preferably, X is a flexible linker as defined above.

Preferably Y is lysine in formula (I).

Preferably Y is glutamic acid in formula (II).

Preferably _(q) is 1 to 5.

Preferably _(n) is 2.

The present invention also provides a fibre-shaping peptide having thesequence: (NH₃-KIRRLKQKNARLK (βA)₃)₂-K

The present invention also provides a fibre-shaping peptide having thesequence: E-((βA)₃ EIAALEYEIAALEQ-COOH)₂

βA as used in the above sequences represents β-alanine.

The present invention also provides a protein structure comprising thefibre-shaping peptide of the present invention.

Preferably the protein structure comprises a plurality of fibre-shapingpeptides according to the present invention and a plurality ofself-assembling peptides as defined above which can self-assemble toform a linear protein structure, wherein the fibre-shaping peptides andthe self-assembling peptides self-assemble to form a protein structure.

As will be appreciated by those skilled in the art, in order to form aprotein structure the plurality for self-assembling peptides willcomprise a first set of self-assembling and a second set ofself-assembly fibres which interact to form a substantially linearstructure. Preferably, the ratio of fibre-shaping peptides: firstself-assembly peptides: second self-assembly peptides comprised in theprotein structure of the present invention is from about 1×10⁻⁶:1:1 to10:1:1, more preferably from about 1×10⁻⁴:1:1 to 2:1:1.

The term “protein structure” refers to any combination of secondaryprotein structures, such as helices and β strands. It is particularlypreferred that the protein structure is or comprises one or more proteinfibres, wherein the protein fibres are as defined above.

Preferably the protein structure of the present invention compriseskinked and waved fibres.

Preferably the protein structure of the present invention comprisessplit and branched fibres.

The present invention also provides a method for producing the proteinstructure of the present invention, comprising mixing a plurality offibre-shaping peptides of the present invention and a plurality ofself-assembling peptides under conditions so that the peptides associateto form a protein structure.

Suitable conditions for forming a protein structure by mixing thepeptides will be apparent to those skilled in the art, especially inview of the information given in WO 01/21646.

The present invention also provides a kit for producing the proteinstructure of the present invention, wherein the kit comprises aplurality of the fibre-shaping peptides of the present invention and aplurality of self-assembling peptides, wherein the fibre-shapingpeptides and the self-assembling peptides can associate to form aprotein structure.

By controlling the amount of fibre-shaping peptides in the proteinstructure, the morphology of the protein structure can be changed.Accordingly, it is possible to have some control over the proteinstructures being generated. In particular, protein fibres can bearranged to form 2 and 3 dimensional assemblies such as grids,scaffolds, filters, networks and matrices. Such protein structures canbe used in a number of applications such as in the purification ofbiological fluids such as blood, or the assembly of cells for cell andtissue engineering purposes. The protein structures may also be used forsurface engineering (see Zhang et al., Biomaterials, 16, 1385-1393,1995).

Furthermore, as the fibre-shaping peptides can comprise functionalmolecules the protein structure can be functionalised. For example, ifthe functional molecules are capable of specifically binding a desiredcomponent or contaminant, the matrix can be used as an affinity matrixfor isolating a desired component or for removing a contaminant. Forexample in the case of virus removal from a blood sample, a binder forthe target contaminant (e.g a peptide or protein with natural orengineered affinities for a viral coat protein) is the functionalmolecule attached to the fibre-shaping peptide. The matrix can then beremoved from blood along with any bound contaminants by lightcentrifugation.

The protein structures of the present application have a number of otherapplications including:

-   -   i. preparation of organised networks for seeding the        crystalisation of biomolecules for X-ray crystallography;    -   ii. using protein structures to promote cell growth for tissue        engineering;    -   iii. the construction of nanoscale molecular sieves and other        devices;    -   iv. the preparation of nanoscale molecular grids/scaffolds that        could be used as supports for a variety of functional molecules;    -   v. functionalised protein structures could be used in, for        example, catalysis, affinity-sieving/purification of biological        fluids and other research solutions, the recruitment of        endogenous molecules and co-factors to promote tissue repair and        tissue engineering in general.

Although the fibre-shaping peptide of the present invention may compriseone or more functional molecules, additional functional molecules can beattached to the protein structure of the present invention at anyappropriate site using standard coupling techniques.

The present invention is now described, by way of example only, withreference to the accompanying Figures.

FIG. 1 illustrates the design principles and sequences for theself-assembling fibre (SAF) and fibre-shaping (FiSh) peptides: a. Thestandard SAF peptide sequences each shown divided into two blocks: A andB for SAF-p1; C and D for SAF-p2a. a & b. Block A complements D and Bcomplements C. This leads to sticky-ended dimers that assemble furtherinto fibres. The register of the assembly is partly maintained by thekey asparagine residues highlighted by the asterisks. c & d, Thediscontinuities designed to be introduced by the FiSh peptides CC^(NN)and DD^(CC), respectively. In b, c & d, the direction of the polypeptidechain (N- to the C-termini) is shown by the arrowheads. This relates tothe nomenclature of the FiSh peptides; for example, CC^(NN) is so-namedbecause it comprises two copies of the block C from SAF-p2a linkedthrough their C-termini, which leaves both N-termini free at the ends ofthe construct. The FiSh peptides are shown kinked rather than straightas the linkers contain flexible b-alanine residues.

FIG. 2 shows uranyl-acetate stained TEM images of fibres formed from theSAF and SAF-FiSh systems. a, straight fibres formed from mixing SAF-p1and SAF-p2a in a 1:1 ratio. b-d, kinked and waved fibres formed byadding the CC^(NN) FiSh peptide to fresh SAF-p1/SAF-p2a mixtures in1:1:1 (b), 0.1:1:1 (c), and <0.1:1:1 (d) (FiSh:SAF-p1:SAF-p2a) ratios.e&f, split and branched (bounded by the box in f) fibres formed byadding the DD^(CC) FiSh peptide to fresh SAF-p1/SAF-p2a mixtures in0.01:1:1 ratios.

FIG. 3 shows the results of an analysis of the morphological changesintroduced into the self-assembling fibres: a, shows the average numberof features (kinks or splits) per 10 μm length of fibres. b, shows theaverage length of the mature fibres as functions of the ratio of FiSh toSAF peptides. Data points for CC^(NN)-containing fibres are shown ascircles, whereas those for DD^(CC)-containing fibres are shown assquares. In panel a the numbers of kinks and splits produced by DD^(CC)are distinguished by solid and broken lines, respectively. Analysis: thenumbers given are averages measured over 80-100 fibres. Standarddeviations on the measurements in a were: 2.6 (for 0.01:1:1) and 0.2(for <0.01:1:1) for the CC^(NN)-containing fibres; 0.4 (for 0.01:1:1)and 0.1 (for <0.01:1:1) for the DD^(CC)-containing split fibres, and 0.4(for 0.01:1:1) and 0.1 for (>0.01:1:1) for the kinked fibres. Standarddeviations on the measurements in b were 5.6 μm (for 0.01:1:1) and 1.6μm (for >0.01:1:1) for data from both FiSh peptides.

FIG. 4 shows transmission electron microscopy images of the originalSAF-p1:SAF-p2-based fibres without (a) and with (b) CC^(NN).

FIG. 5 shows a transmission electron microscopy image of a straightpeptide fibre matrix having recruited gold peptides via streptavidin,which in turn was recruited to the fibres by biotin incorporated intothe self-assembling peptides.

FIG. 6 shows a transmission electron microscopy image of a singlestraight peptide fibre having recruited gold peptides via streptavidin,which in turn was recruited to the fibres by biotin incorporated intothe self-assembling peptides.

FIG. 7 shows a high magnification transmission electron microscopy imageof a single straight peptide fibre having recruited gold peptides viastreptavidin, which in turn was recruited to the fibres by biotinincorporated into the self-assembling peptides.

FIG. 8 shows a transmission electron microscopy image of a kinkedpeptide fibre having a gold particle specifically recruited at the hubof the particle.

EXAMPLES

Materials and Methods

Peptide Synthesis

Peptides were synthesized using standard solid-phase Fmoc chemistry,purified by RP-HPLC and confirmed by MALDI-TOF mass spectrometry.

Fibre Assembly

All samples of fibres were prepared with SAF-p1 and SAF-p2a (each at 100μM concentration) with the designated amounts of FiSh peptides, andincubated at 22° C. overnight following the techniques described inPandya et al., 2000 (supra).

Electron Microscopy

Fibre suspensions were dried onto carbon grids and stained with uranylacetate for electron microscopy as described previously in Pandya etal., 2000 (supra).

Recruitment of Gold Particles

Streptavidin nanogold conjugate (streptavidin labeled with colloidalgold nanoparticles (5 or 10 nm)) were obtained from SIGMA. All thepeptide synthesis reagents including biotinylated Fmoc-lysine werepurchased from Merck Biosciences (Novabiochem). Peptides weresynthesized on a Pioneer Peptide Synthesis System using standardFmoc-chemistry as described above.

The streptavidin nanogold conjugate (SNC) was 2-10 times diluted with 10mM MOPS, pH 7 as the diluent buffer containing 0.05% TWEEN 20 tominimise background. The diluted conjugate was allowed to equilibratefor 30 min in this lower glycerol content at room temperature. Theoptimal concentration was determined empirically (in accordance with theprocedure recommended by SIGMA) to be A₅₂₀=0.25 with incubation time30-45 minutes.

Fibre samples were prepared as indicated above (except biotinylatedFmoc-lysine was used to incorporate biotin into the peptides). Acommercial Fmoc-lysine which had biotin attached the side-chain(epsilon) amino group was used to biotin into the synthesis of bothstraight and fibre shaping peptides. For straight peptides standardlinear synthesis was used. Fibre shaping peptides incorporating biotinwere created using a di-lysine hub, that is Lys-Lys (biotin). The firstlysine acted as the hub from which the two peptide arms were grown. Thealpha and epsilon amino acid groups of this lysine were used to initiatepeptide synthesis. The alpha carboxy group of the first lysine wascoupled to the second lysine, which contained the biotin. A designatedamount (2 μL) of SNC was then added to the fibre preparations. Toachieve better coverage of fibre surfaces with SNC in some applicationseither higher volumes (up to 20 μL) or concentrations (up to 2 timesdilution) were used.

After incubation an 8 μL drop of peptide solution was applied to acarbon-coated copper specimen grid (Agar Scientific Ltd) and dried withfilter paper followed by washing two-three times (3-5 min each) withstandard MOPS buffer to eliminate unspecifically bound SNC. The grid wasstained with filtered 0.5% aqueous uranyl acetate for electronmicroscopy at 20° C.

The novel protein structures described herein were made by combiningSAF-p1 and SAF-p2a in the presence of fibre-shaping peptides based onthe SAF-p2a sequence following the technique described in Pandya et al.,2000 (supra).

The present invention is demonstrated, by way of example only, with twonovel peptides, CC^(NN) and DD^(CC), which introduce kinks/waves andsplits/branches into the SAF fibres, respectively.

The design principles for CC^(NN) and DD^(CC) peptides are shownschematically in FIGS. 1 c&d. The peptides were based on the SAF-p2asequence, FIG. 1 a. For example, in CC^(NN) the N-terminal “C” subunitof SAF-p2a was duplicated in a tail-to-tail fashion; i.e., two copies ofthe C sequence (hence the “CC” term) were linked through their C-terminileaving the N-termini free at the ends of the construct (hence the “NN”superscript). This was achieved by synthesising two C subunitssimultaneously from the two amino groups of a single, bifunctional hub,namely L-lysine attached via its carboxy terminus to a solid-phasepeptide synthesis resin. To allow additional flexibility at the lysinejoint, three β-alanine monomers were added to the amino groups of thelysine prior to synthesis of the C sequences. As depicted in FIG. 1 c,this should set up the possibility for CC^(NN) to interact with twocopies of SAF-p1. However, this interaction is altogether different fromthe interaction originally prescribed for the self-assembling peptides(straights), SAF-p2a and SAF-p1: the association of these is paralleland contiguous, FIGS. 1 a&b, which leads to linear and potentiallyinfinite assemblies (Pandya et al., Biochemistry, 39, 8728-8734, 2000);in contrast, the addition of CC^(NN) aimed to force two neighbouringSAF-p1 peptides to converge; the reason for including the β-alaninelinker was thus to accommodate any resulting discontinuity and, so,effectively allow fibrillogensis from the two SAF-p1 peptides, FIG. 1 c.Similar principles were used in the DD^(CC) design except that twocopies of the D subunit of SAF-p2a were linked through their N-terminiusing L-glutamic acid as a hub, FIG. 1 d; again three β-alanine residueswere used to separate each of the D peptide sequences from the hub.There are subtle differences in the sequences of the C and D units, FIG.1 a, which, as discussed below, resulted in different morphologicalchanges in the SAF assemblies.

Consistent with the design, when CC^(NN) was doped into a freshSAF-p1/SAF-p2a mixture the resulting matured fibres were not straight,but kinked or wavy, FIGS. 2 b,c&d. Varying the ratio of CC^(NN) in thestarting mixtures altered the numbers of kinks per unit length offibres, FIG. 3 a. However, this was at the expense of fibre integrity:the inclusion of more CC^(NN) reduced the length of the fibres that wereformed, FIG. 3 b. Though some linear fibres were observed these wererare, and the number reduced rapidly with increasing amounts of CC^(NN).

Though these observations are consistent with the design of the CC^(NN)FiSh peptide, it is perhaps surprising that the kinked fibres appear sorigid. It is probable that the aforementioned thickening of the fibresstabilizes them and thus limits kinking. To test this, we preparedsamples of the original SAF-p1:SAF-p2 design without CC^(NN); note thatthe coiled-coil interfaces of original and redesigned SAF peptides andthe FiSh peptides were unaltered, and so remained compatible. Thesequence of SAF-p2 is KIALKAKNAHLLKQEIAALEQEIAALEQ, which differs atfour residues from SAF-p2a. SAF-p2 combines with SAF-p1 to give fibresthat are approximately two thirds the diameter of the redesignedSAF-p1:SAF-p2a fibres, and are less stable to heat (A. M. Smith & D. N.Woolfson, unpublished results). Samples were prepared by incubatingSAF-p1 and SAF-p2 with or without CC^(NN) (each peptide was at 100 μMconcentration) at 5° C. for 1 hour before the standard preparation forelectron microscopy. Without CC^(NN), SAF-p1+SAF-p2 produced extendedlinear fibres, FIG. 4 a, but with the FiSh peptide multiple kinks wereapparent, FIG. 4 b. The extent and frequency of kinking in theSAF-p1:SAF-p2 background was greater than that observed withSAF-p1+SAF-p2a. This suggests that the flexibility of the SAF plays arole in kinking: the thinner, less-stable SAF-p1:SAF-p2 fibres containmore kinks. Consistent with this, the fibre shortening observed for FiShpeptides in the redesigned SAF-p1:SAF-p2a background, FIG. 3 b, was lessapparent in the original SAF-p1:SAF-p2 background, FIG. 4.

Intriguingly, inclusion of DD^(CC) in fresh SAF-p1/SAF-p2a mixtures ledto two different morphologies in the matured fibres: with small amountsof the FiSh peptide (101:1:1 of DD^(CC):SAF-p1:SAF-p2a) the fibreskinked as observed with CC^(NN). However, as the ratio of DD_(CC) wasincreased (up to 1:1:1) less kinking was observed, and instead thefibres tended to split or branch, FIGS. 2 e&f and FIG. 3 a.

The difference in behaviour of the two FiSh peptides must result fromthe different sequences of the C and the D subunits, FIG. 1 a. In theoriginal SAF design, the C subunit of SAF-p2 was made to partner the Bsubunit of SAF-p1 specifically by the inclusion of complementaryasparagine residues in each of the peptides at a key interfacial site(Pandya et al., Biochemistry, 39, 8728-8734, 2000); this feature waspreserved in the SAF-p2a design. This is a so-called negative-designprinciple (Beasley et al., J. Biol. Chem., 277, 10150-10155, 2002); thatis, it is a feature incorporated to direct the assembly of one structureand guard against the formation of potential alternatives. In the caseof the SAFs, the asparagine residues were included to ensure parallelheterodimer formation (Harbury et al., Science, 262, 1401-1407, 1993,O'Shea et al., Curr. Biol., 3, 658-667, 1993, Woolfson et al., Prot.Sci., 4, 1596-1607, 1995, O'Shea et al., Science, 254, 539-44, 1991,Lumb et al., Biochemistry, 34, 8642-8648, 1995 and Gonzalez et al.,Nature Struct. Biol., 3, 1011-1018, 1996) to offset the register of thetwo peptides and hence to promote fibrillogenesis (Pandya et al.,Biochemistry, 33, 8728-8734, 2000). Although the inclusion of asparagineincreases dimer and register specificity in leucine-zipper peptides thisis at the expense of overall stability (Harbury et al., Science, 262,1401-1407, 1993, Lumb et al., Biochemistry, 34L 8642-8648, 1995 andGonzalez et al., Nature Struct. Biol., 3, 1011-1018, 1996). The CC^(NN)peptide has two such asparagines and the DD^(CC) has none. Therefore,the inventors suspect that the DD^(CC) forms stronger leucine-zipperinteractions and is potentially the more promiscuous in its interactionswith the standard SAF peptides, and that this leads to split and/orthickened appearance of some of the fibres, FIG. 2 e&f. Put another way,CC^(NN) is more selective in the interactions it makes. This, combinedwith the presumed lower stability of B:C interactions compared with A:Dinteractions, leads to fewer and more-easily rectified imperfections(such as splitting and thickening).

Following the scheme of FIG. 1, there are sixteen possible combinationsof the C and D subunits of SAF-p2a. We synthesised two of these, CD^(NC)and CC^(NC), as control peptides. CD^(NC) was simply the SAF-p2asequence with a spacer inserted between the C and D subunits; in CC^(NC)two successive copies of C were separated by the spacer; in both casesthe spacer comprised three β-alanine residues, a central ε-aminohexanoicacid residue (as a substitute for the L-lysine and L-glutamic acid hubsused in CC^(NN) and DD^(CC)), followed by three further B-alanineresidues. Neither CD^(NC) nor CC^(NC) caused kinking, waving, splittingor branching of the matured fibres as observed for CC^(NN) and DD^(CC).However, at ratios of 10⁻²:1:1 (control:SAF-p1:SAF-p2a) and above, bothCD^(NC) and CC^(NC) inhibited fibre assembly completely; mixtures withsmaller amounts of control peptide did produce fibres, but these wereshort (<20 mm) and rare (500 times less abundant than in mixturescontaining the FiSh peptides CC^(NN) and DD^(CC)). In other words,CD^(NC) and CC^(NC) acted as terminators in fibrillogenesis.

In summary, the inventors have presented experimental data for alteringthe shapes of designed self-assembly fibres that originally formedexclusively linear and non-branched structures. Fibre-shaping (FiSh)peptides were added to mixtures of peptides that would otherwise haveformed linear assemblies. The two FiSh peptides tested influenced fibresmorphology differently: one kinked the fibres, whereas the other split,or branched them. The ratio of FiSh to standards peptide determined thenumber of kinked and branched features observed.

The ability to alter fibre morphologies can be used in the developmentof biomaterials that respond to cues provided by their environment. Suchcues might be presented as a pattern on a surface. This could lead tosurfaces functionalised with proteins for the applications inprotein-array technology and the development of new protein-baseddiagnostic/sensor devices. Another possibility is for the assembly ofnetworks that might be used as scaffolds in cell and tissue engineering.In this case the self-assembly fibres could be induced to respond to,and so support cell growth.

As indicated above, as the FiSh peptides may comprise other functionalmolecules. In this way the FiSh peptides could be used to recruitbioactive peptides, proteins and small molecules to assembled fibres.For example, the additional moiety could be a peptide antigen, whichonce incorporated into the fibre could be used to pull-down (i.e.recruit) a specific antibody to the fibre surface. Alternatively, if thefunctional molecule is a nucleic acid sequence, transcription factorsthat interact with the nucleic acid sequence may be isolated. In theserespects, the FiSh peptides should be considered as nodes at whichfunctional groups could be located.

10 nm gold particles were recruited to both straight fibres and thekinked fibres of the present invention using standardbiotin/streptavidin chemistry as described above. In particular, biotinwas incorporated into the self-assembling peptides during synthesis andthe gold particles were coated with streptavidin. It was found that thegold particles were randomly distributed on the straight fibres (seeFIGS. 5 to 7) but were specifically recruited at the hub of the kinkedfibres (see FIG. 8).

In the straight fibres one of the amino acids in the “f” position of theheptad was derivatised with biotin. In the kinked fibre the hub wasderivatised with biotin. It is advantageous to be able to specificallyrecruit functional molecules such as gold particles to specific siteswithin the kinked fibres of the present invention.

All documents cited above, are incorporated herein by reference.

1. A fibre-shaping peptide comprising a hub and a plurality of peptidemonomer units each being attached at one end thereof to the hub, whereinthe free ends of at least 2 peptide monomer units are N-termini orC-termini, and each of the at least 2 peptide monomer units is capableof interacting with a sub-unit of a self-assembling peptide to form anoverlapping staggered structure.
 2. The fibre-shaping peptide accordingto claim 1, wherein the hub is one or more amino acids residue having aplurality of derivatisable sites to which the peptide monomer units canbe attached.
 3. The fibre-shaping peptide according to claim 2, whereinthe hub is lysine or glutamic acid.
 4. The fibre-shaping peptideaccording to claim 1, which comprises 2 to 4 peptide monomer units. 5.The fibre-shaping peptide according to claim 1, which comprises 2peptide monomer units.
 6. The fibre-shaping peptide according to claim1, wherein each peptide monomer unit is attached to the hub via aflexible linker.
 7. The fibre-shaping peptide according to claim 6,wherein the flexible linker is a peptide linker comprises amino acidsselected from the group consisting of glycine, alanine, serine andβ-alanine.
 8. The fibre-shaping peptide according to claim 6, whereinthe flexible linker is a poly-β-alanine peptide.
 9. The fibre-shapingpeptide according to claim 1 which additionally comprises one or morefunctional molecules attached to the hub.
 10. The fibre-shaping peptideaccording to claim 9, wherein the functional molecule is an antibodymolecule, a receptor, a ligand, an enzyme, an antigen, a label, a metalion or a nucleic acid molecule.
 11. The fibre-shaping peptide accordingto claim 9, wherein the functional molecule is attached to the hub via aflexible linker.
 12. The fibre-shaping peptide according to claim 11,wherein the flexible linker is a peptide linker comprises amino acidsselected from the group consisting of glycine, alanine, serine andβ-alanine.
 13. The fibre-shaping peptide according to claim 11, whereinthe flexible linker is a poly-β-alanine peptide.
 14. The fibre-shapingpeptide according to claim 1, wherein the at least 2 peptide monomerunits comprise a heptad repeat motif and/or a hendecad repeat motif. 15.The fibre-shaping peptide according to claim 14, which is capable offorming a staggered parallel coiled coil structure with aself-assembling peptide which comprises a heptad repeat motif and/or ahendecad repeat motif.
 16. The fibre-shaping peptide according to claim1 having the formula:(NH₃-g(abcdefg)_(q)abcde-(X)_(m))_(n)—Y—((X)_(m)-Z)_(p)  (I)or(Z-(X)_(m))_(p)—Y—((X)_(m)-g(abcdefg)_(q)abcdef-COOH)_(n)  (II) whereinabcdefg is a heptad repeat motif; X is a flexible linker; Y is a hub; Zis a functional molecule; _(q) is 2 to 15; _(m) is 0 or 1; _(n) is 2 to4; and _(p) is 1 to
 4. 17. The fibre-shaping peptide according to claim16, wherein X is a poly-β-alanine peptide.
 18. The fibre-shaping peptideaccording to claim 16, which has the formula designated as I and whereinY is lysine.
 19. The fibre-shaping peptide according to claim 16, whichhas the formula designated as II and wherein Y is glutamic acid.
 20. Thefibre-shaping peptide according to claim 16, wherein _(q) is 1 to
 5. 21.The fibre-shaping peptide according to claim 16, wherein _(n) is
 2. 22.A fibre-shaping peptide having the sequence: (NH₃-KIRRLKQKNARLK(βA)₃)₂-K.
 23. A fibre-shaping peptide having the sequence: E-((βA)₃EIAALEYEIAALEQ-COOH)₂


24. A protein structure comprising a fibre-shaping peptide according toclaim
 1. 25. A protein structure comprising a plurality of fibre-shapingpeptides according to claim 1 and a plurality of self-assemblingpeptides, wherein the first and second peptide monomers self-assemble toform a non-linear protein structure.
 26. The protein structure accordingto claim 23, wherein the ratio of fibre-shaping peptides: firstself-assembling peptides: second self-assembling peptides is from about1×10⁻⁶:1:1 to 10:1:1.
 27. The protein structure according to claim 25,which comprises kinked and waved protein fibres.
 28. The proteinstructure according to claim 25, which comprises split and branchedprotein fibres.
 29. The protein structure according to claim 25, whichis a matrix, a grid, a scaffold, a filter or a network.
 30. The proteinstructure according to claim 25, wherein the plurality ofself-assembling peptides comprises peptides having the sequenceNH₃-KIAALKQKIASLKQEIDALEYENDALEQ-COOH and peptides having the sequenceNH₃-IRRLKQKNARLKQEIAALEYEIAALEQ-COOH.
 31. A method of producing aprotein structure comprising a plurality of fibre-shaping peptidesaccording to claim 1 and a plurality of self-assembling peptides,wherein the first and second peptide monomers self-assemble to form anon-linear protein structure, comprising mixing a plurality offibre-shaping peptides according to claim 1 and a plurality ofself-assembling peptides under conditions so that the peptides associateto form a protein structure.
 32. A kit for producing a proteinstructure, wherein the kit comprises a plurality of fibre-shapingpeptides according to claim 1 and a plurality of self-assemblingpeptides which, wherein the first and second peptide monomers canassociate to form a protein structure.
 33. (canceled)
 34. (canceled) 35.(canceled)
 36. A self-assembling peptide having the sequenceNH₃-KIRRLKQKNARLKQEIAALEYEIAALEQ-COOH (SAF-p2a).